Abstract PD10-10: PD10-10 Single cell characterization of longitudinal biopsies from breast cancer patients treated with the aromatase inhibitors letrozole and exemestane in sequence
Authors: Salim Ghannoum, Chloé Steen, Marie Fongård, Marius Bjørnstad, Laurens Reitsma, Stephanie Geisler, Manouchehr Seyedzadeh, Unn-Cathrin Buvarp, Marie Loeng, Torben Lüders, Diether Lambrechts, Marianne Lyngra, Vessela Kristensen, Jürgen Geisler, Xavier Tekpli
Journal: Cancer Research (2023)

Abstract Background About 70% of breast cancer cases are hormone receptor positive, indicating that cancer cells exploit estrogens for their growth. Postmenopausal estrogen receptor positive breast cancer patients are currently often treated with aromatase inhibitors suppressing serum and tumor tissue estradiol levels by &amp;gt;90%. Two widely used aromatase inhibitors are letrozole, a nonsteroidal inhibitor and exemestane, a steroidal aromatase inactivator. While the mechanisms of action of these two drugs are well studied, their effects on the tumor immune microenvironment and mechanisms of resistance to these drugs are still not sufficiently elucidated. Study design The NEOLETEXE trial1 was a neoadjuvant, randomized, open-label, intra-patient, cross-over, single center clinical trial aiming at treating postmenopausal patients with locally advanced breast cancer defined primarily as large T3/T4 and or N2/N3. However, patients with large T2 tumors were also eligible. Patients were randomized to neoadjuvant therapy with either letrozole (2.5 mg daily) or exemestane 25 mg daily for about 3 months, followed by a cross-over to the alternative drug for another 3 months prior to surgery. 102 patients were enrolled in NEOLETEXE. Methods Pre-treatment, on-treatment (3 months) and end-of-treatment (6 months of therapy) biopsies were subjected to single-cell transcriptome, T cell receptor and B cell receptor profiling using the Chromium Single-Cell v2 5′ Chemistry (10x Genomics). Libraries were paired-end sequenced on a NovaSeq6000. Single cell gene expression matrices were analyzed with the Seurat package (v4.0.2). After filtering out stressed/dying cells or cells with low quality sequencing, gene expression of the remaining good quality cells was normalized and scaled to construct principal components and further cluster cells. Results We clustered 362.762 cells from 26 pre-treatment, 20 on-treatment and 19 end-of-treatment biopsies and identified 8 main cell types to be present: T cells, B cells, epithelial cells, fibroblasts, endothelial cells, macrophages, mast cells and dendritic cells. To further identify specific and specialized cell subtypes, we clustered the cells belonging to the above-mentioned cell types independently and annotated the clusters obtained using validated marker genes. Finally, we use statistical methods and algorithms to characterize how the proportion of the different cell types changes in tumors under treatment pressure. Specifically, we show changes in the proportion of CD8 effector memory cells under treatment pressure, with a significant increase in cytotoxic T cell proportions after two months of treatment with aromatase inhibitor. Conclusions We use single cell profiling to obtain a high-resolution map of the cell types found in tumor biopsies of the NEOLETEXE trial to characterize the effects of aromatase inhibitors on the tumor microenvironment and to identify the cancer cell signatures during treatment with letrozole or exemestane. 1. Bahrami N., Sauer T., Engebretsen S., Aljabri B., Bemanian V.,Lindstrøm J., Lüders T., Kristensen V.N., Lorentzen A., Loeng M., Ødegård H.P., Kvaløy J.Ø., Vestøl I.B., Geisler S.B., Gravdehaug B., Gundersen J.M., Geisler J. The NEOLETEXE trial: a neoadjuvant cross-over study exploring the lack of cross resistance between aromatase inhibitors. Future Oncology, 15 (32), 3675-3682, 2019. Citation Format: Salim Ghannoum, Chloé Steen, Marie Fongård, Marius Bjørnstad, Laurens Reitsma, Stephanie Geisler, Manouchehr Seyedzadeh, Unn-Cathrin Buvarp, Marie Loeng, Torben Lüders, Diether Lambrechts, Marianne Lyngra, Vessela Kristensen, Jürgen Geisler, Xavier Tekpli. PD10-10 Single cell characterization of longitudinal biopsies from breast cancer patients treated with the aromatase inhibitors letrozole and exemestane in sequence [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD10-10.